INTRODUCTION

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Aromatic dicationic compounds, including bisbenzamidines and dicationically substituted bisbenzimidazoles and carbazoles, have excellent experimental anti-Pneumocystis activities (14, 40, 46, 48, 50, 51) and are also active against other microbial pathogens, including protozoan parasites (2-5, 10, 38, 41, 43, 44), fungi (45), and some viruses (25-27, 49, 53). Aromatic dications also possess other pharmacological properties, including antiinflammatory and anticoagulant activities (29-37). Two problems hindering development of these compounds as new drugs, however, are limited oral bioavailability and toxicity (24, 38, 48, 51).

Recent studies of pentamidine metabolism (7-9, 21-23) have led to a novel approach to overcome the limited oral bioavailability and acute toxicity. Aromatic diamidoximes are hypothesized to be orally bioavailable prodrugs that are readily reduced by drug-metabolizing enzymes to the active aromatic amidines (19, 21, 22), resulting in excellent antimicrobial activity with reduced acute host toxicity.

Amidoximes were first shown by Lamb and White to be active against experimental African trypanosomiasis (42) and then later were shown to be active against other microorganisms (1, 17, 18, 28). Although activities were often reported for both amidoximes and corresponding amidines, no mention was made in these early publications that metabolic activation was required for in vivo activity of the amidoximes. Moreover, no systematic studies were performed to determine which analogs were orally active and if the amidoxime derivatives had increased oral activity compared to the amidines. Thus, the concept of amidoximes as prodrugs of amidines was not raised in earlier studies.

The hypothesis that amidoximes might be useful prodrugs resulted from research examining the metabolism of pentamidine (6-9, 21, 22). Two primary oxidative metabolites identified were the mono- and diamidoximes, formed by N-hydroxylation of pentamidine. Although the diamidoxime derivative of pentamidine has little or no activity against three protozoan parasites in vitro, both the mono- and diamidoximes were active against African trypanosomes and Leishmania spp. when given to experimental animals subcutaneously (19, 21-23, 39). The diamidoxime given orally to rats was absorbed from the gut and converted to pentamidine, a reaction subsequently shown to be catalyzed by an oxygen-independent hepatic reductase activity (21, 22). These observations led to the proposal that amidoxime derivatives, in general, are effective, orally absorbed prodrugs for all pharmacologically active amidine-containing compounds (19). However, the only amidoximes tested were derivatives of pentamidine. We recently demonstrated that two novel amidoximes of 2,5-bis[4-amidinophenyl]furan were highly active orally and intravenously (13). Moreover, Weller and coworkers demonstrated that amidoximes of potent monoamidine fibrinogen receptor antagonists greatly enhanced their oral bioavailability (54).

With this promising background, we began to synthesize potential amidoxime prodrugs of our most active, least toxic diamidines. Results presented here, however, demonstrate that amidoximes are not effective prodrugs for all aromatic dicationic compounds. The nature of the linker between the two amidoxime moieties plays a key role in determining if a particular diamidoxime has oral anti-Pneumocystis activity. Diamidoxime derivatives of the very promising bisbenzimidazole and carbazole classes of dications, and bisbenzamidoximes that contain additional nitrogen atoms in the aliphatic linkers, had little or no anti-Pneumocystis activity, even though the parent diamidines had excellent intravenous activity. Variability in activity does not appear to be caused by differences in enzymatic reductase activity, since both active and inactive diamidoximes were metabolized by cell-free liver homogenates.

	MATERIALS AND METHODS

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Synthesis of compounds. The known diamidine compounds tested in this study were synthesized in our laboratory by previously described methods (7, 40, 48, 51). The novel diamidines 12, 14, 18, and 19 (Table 1) were prepared by similar methods. The known di- and monoamidoximes (compounds 1 and 9 [Table 1]) of pentamidine were prepared according to the procedures of Clement and Raether (23). All novel amidoximes were prepared by similar procedures, with the exception of compound 19, which was prepared by a Pinner synthesis from the corresponding dinitriles. Each compound was characterized by high-performance liquid chromatography (HPLC), elemental analysis, high-resolution fast atom bombardment mass spectrometry, and proton magnetic resonance. Structures of test compounds are shown in Table 1, and the melting points and elemental analyses for the novel compounds are given in Table 2.

Anti-Pneumocystis activity. The induction and treatment of Pneumocystis pneumonia in the rat model were carried out according to published methods (40, 50, 51). Adult male Sprague-Dawley rats, barrier raised, not certified virus free, and weighing 150 to 200 g, were obtained from Hilltop Laboratories (Scottdale, Pa.). The individually caged animals were begun immediately upon arrival on an immunosuppressive regimen consisting of a low-protein (8%) diet (Zeigler Brothers, Gardner, Pa.) and drinking water containing tetracycline (0.5 mg/ml) and dexamethasone (1.0 µg/ml). This regimen was continued for the next 8 weeks, with animals monitored daily and weighed weekly. At the beginning of the 7th week, animals were divided into groups of at least six animals per group and the test compounds were administered daily for 14 days either orally by gavage or intravenously by tail vein injection. Compounds were routinely tested orally at 33 µmol kg of body weight¹ day¹ and intravenously at 22 µmol kg of body weight¹ day¹. Saline- and pentamidine-treated groups were included as negative and positive controls, respectively.

In vivo toxicities of test compounds. Preliminary evaluations of relative toxicities of test compounds were performed in two ways. First, the dexamethasone-immunosuppressed test rats were closely observed throughout the 14 days of intravenous or oral administration for overt toxic responses. Animals were observed closely for a 10- to 15-min period following injection of the test drug each day for signs of acute toxicity, including the hypotensive response (paling of eyes and paws, dyspnea, lethargy, and decreased body temperature) elicited by intravenous pentamidine at its effective dose. Their overall health and general well-being were observed and recorded on a daily basis for the remainder of the experiment. Excessive weight loss (more than a twofold loss compared to the saline controls over the 2-week dosing period) was considered a key indicator of declining health due to drug toxicity. At necropsy, the liver, spleen, kidneys, and pancreas were removed from each animal and examined for gross pathology. Subjective scores (40, 51) of toxicity associated with multiple dosing were assigned to each compound (Table 1) and are discussed further in Results and Discussion.

In vitro metabolism. In vitro metabolism of diamidoximes by rat liver homogenate 9,000 × g supernatants, postmitochondrial 105,000 × g supernatants, or microsomal fractions was performed as previously described (8, 9). Briefly, adult male barrier-raised Sprague Dawley rats (Hilltop Laboratories) were allowed free access to rat chow (22.5% protein, 5.5% fat, and 4.5% fiber, with essential vitamins and minerals; Agway) and tap water. Rats were euthanized by decapitation, and the livers were removed immediately, rinsed with 50 mM potassium phosphate buffer (pH 7.4), and placed on ice. All subsequent steps were performed at 4°C. The livers were minced and homogenized, and 9,000 × g supernatants, 105,000 × g supernatants, or 105,000 × g microsomal pellets were prepared as described elsewhere (6). Each fraction was assayed for protein content (15) and stored at 80°C. Fractions from rat kidneys, lungs, hearts, and brains were prepared in a similar fashion, as previously described (8, 9).

	RESULTS AND DISCUSSION

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Anti-Pneumocystis activities of bisbenzamidoximes. A major limitation of aromatic dicationic compounds as antimicrobial agents has been their lack of oral activity. Results from the present study demonstrate that four bisbenzamidoximes, diamidoxime derivatives of pentamidine and three novel direct pentamidine analogs, have excellent oral anti-Pneumocystis activities in the immunosuppressed-rat model of disease. Compound structures and anti-Pneumocystis activities of these bisbenzamidoximes and their corresponding bisbenzamidines are given in Table 1.

In agreement with previous results (19), the diamidoxime derivative (compound 1) of pentamidine had significant anti-Pneumocystis activity when given orally by gavage at 33 µmol kg¹ day¹, compared to the oral saline control group (Table 1). The novel bisbenzamidoxime compounds 3, 5, and 7 were even more active, with the lowest cyst scores reported to date from our laboratories for orally administered aromatic dicationic compounds (40, 48, 51).

In contrast, the corresponding diamidines were less active when given orally (Table 1). Pentamidine (compound 2) was completely inactive at the oral daily dose of 33 µmol kg¹ for 14 days. Cyst scres for diamidine compounds 4 and 6 were slightly, but not significantly, reduced compared to the oral saline controls. Cyst counts were more than 15-fold higher for these diamidines than for their corresponding diamidoximes, compounds 3 and 5 (Table 1). The only diamidine compound with good oral activity was compound 8. Its diamidoxime analog, compound 7, had a slightly lower mean cyst score.

Each of the bisbenzamidoxime compounds 1, 3, 5, and 7 also had excellent anti-Pneumocystis activity when given intravenously at 22 µmol kg¹ once daily for 14 days (Table 1). Mean cyst counts were greatly reduced compared to those for the saline controls and were also significantly lower than the mean count for the intravenous pentamidine (compound 2) control group. The diamidine compounds 4, 6, and 8 have previously been shown to have intravenous activity (40, 48; Table 1). Direct comparisons of intravenous anti-Pneumocystis activities of these diamidines to those of the corresponding diamidoximes, compounds 3, 5, and 7, cannot be made because the intravenous activities of diamidines were previously evaluated by a different cyst score method and at slightly different doses. Subjectively, however, diamidoximes do appear to compare favorably with the corresponding diamidines with regard to intravenous efficacy.

Anti-Pneumocystis activities of monobenzamidoximes. Substitution of amidoxime moieties for both dicationic groups may be required for oral but not for intravenous anti-Pneumocystis activity. Two mono-substituted amidoximes were synthesized and tested (Table 1). Compound 9, the monoamidoxime analog of pentamidine, was significantly active at an intravenous dose of 22 µmol kg¹ day¹. The compound was inactive at an oral dose of 33 µmol kg¹ day¹, however. A second monoamidoxime, compound 10, which contains one amidoxime moiety and one imidazoline cationic group, was active intravenously at a dose of 10 µmol kg¹ day¹ but was not active when given orally at the high dose of 58 µmol kg¹ day¹. Thus, both cationic moieties apparently must be in the amidoxime form for oral uptake to occur.

Diamidoximes that lack activity against Pneumocystis. Not all diamidoxime compounds have improved antimicrobial activity compared to the diamidine analogs, indicating that the prodrug approach will not work for all classes of aromatic dicationic compounds. The potent antitrypanosomal diamidine compound, diminazine (Berenil), has excellent in vivo activity against African trypanosomes and Leishmania; its diamidoxime derivative, however, was only marginally active when given subcutaneously (20). Diminazine has a triazine bridge connecting the benzamidino moieties. Our current results indicate that diamidoximes from three other classes of compounds lack oral and intravenous activity against Pneumocystis. These diamidoximes, like the diamidoxime of diminazine, also have nitrogen atoms in positions other than the amidoxime moieties and lack the ether oxygens in the bridge between aromatic groups.

(i) Bisbenzamidoximes that lack anti-Pneumocystis activity. Two novel bisbenzamidine compounds (compound 12 and 14) (Table 1), each containing internal amide groups as part of the bridge linking the benzamidine moieties, were synthesized and tested for activity against Pneumocystis. The compounds were synthesized because they were hypothesized to have improved DNA binding properties, increased aqueous solubility, and reduced metabolism along the cleavage pathways that have been proposed to decrease the activity and increase the toxicity of pentamidine. Both diamidines had significant anti-Pneumocystis activity when administered at 22 µmol kg¹ day¹ intravenously. The diamidoxime derivatives (compounds 11 and 13) of each compound, however, were not active, either intravenously (22 µmol kg¹ day¹) or orally (33 µmol kg¹ day¹) (Table 1).

(ii) Bisbenzimidazole and carbazole diamidoximes that lack anti-Pneumocystis activity. Dicationically substituted bisbenzimidazoles have excellent activity against Pneumocystis, with selected compounds showing marked improvements in toxicity and pharmacologic properties compared to pentamidine (51). However, oral bioavailability for these compounds also appears to be limited. To determine if the prodrug approach could be used for this class of aromatic dications, we synthesized and tested the diamidoxime derivative (compound 15) (Table 1) of one of our most active bisbenzimidazole diamidines (compound 16). Although the diamidine has been shown to have excellent anti-Pneumocystis activity at 18 µmol kg¹ day¹ intravenously, the diamidoxime was completely inactive intravenously and orally (Table 1). One other bisbenzimidazole diamidoxime, in which the benzimidazole nitrogen was substituted with a methyl group (compound 17) (Table 2), was synthesized and tested. The diamidine analog (compound 18) had excellent intravenous activity at 22 µmol kg¹ day¹. The diamidoxime, however, lacked significant intravenous and oral activity (Table 1).

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Toxicity of bisbenzamidoximes. A second major factor limiting development of aromatic dications as antimicrobial drugs has been toxicity. We report here preliminary observations of acute and subchronic toxicity in rats treated intravenously and orally with three bisbenzamidoximes active against Pneumocystis. Although the toxicity information presented is mainly anecdotal and should not be considered definitive, these evaluations do permit important preliminary comparisons of relative toxicities of compounds.

(i) Acute toxicity in nonimmunosuppressed rats. A preliminary dose escalation study was performed with non-dexamethasone-treated rats to compare overt acute toxic responses elicited by diamidoxime compounds 1, 5, and 7 and their corresponding diamidines. Compound 3 was not available in sufficient quantity for evaluation of acute toxicity. Overt acute adverse reactions following single intravenous bolus injections were greatly reduced for the three bisbenzamidoximes compared to those for the bisbenzamidines, and no adverse reactions were observed after high single oral doses of the bisbenzamidoximes.

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(ii) Subchronic toxicity in corticosteroid-immunosuppressed rats. Although the bisbenzamidoxime compounds 1, 5, and 7 appear to cause less overt acute toxicity following single intravenous injections, the diamidoximes do retain substantial overt toxicity in the dexamethasone-suppressed rats treated with test compounds for 14 days. Subjective scores of multiple dosing toxicity were assigned and are included in Table 1. Pentamidine given at its intravenous therapeutic dose of 22 µmol kg¹ day¹ has been assigned a subjective toxicity score of ++, primarily because of its hypotensive response but also because it can cause inflammation of the tail with multiple injections. The four bisbenzamidoximes (compounds 1, 3, 5, and 7) tested were assigned scores of + to +++ when given intravenously. Each bisbenzamidoxime caused substantial inflammation of the tail. The tails of most animals in each group became reddened and swollen after 3 to 5 days of injections; then many became severely necrotic before the end of the 14-day experiment. The response to compound 3 was particularly severe. In addition, diamidoxime compound 7 caused deaths late in the treatment regime. Three of six animals treated intravenously with compound 7 at 22 µmol kg¹ died late (days 11 and 12) during treatment. Animals that died became very pale 2 to 3 days before death, and internal organs were blanched and grossly necrotic upon necropsy. Finally, one animal treated orally at 33 µmol kg¹ day¹ with diamidoxime compound 3 and one treated orally with compound 5 died during the experiment. No other adverse reactions were detected in orally treated animals, and the deaths may not have been treatment related.

Metabolism of amidoximes. (i) Metabolism of bisbenzamidoximes active against Pneumocystis. Data presented in Fig. 1A confirm previous observations that rat liver homogenates reduce the diamidoxime (compound 1) of pentamidine, forming the monoamidine-monoamidoxime (compound 9) and smaller quantities of the diamidine, pentamidine (compound 2). The metabolites have previously been identified by mass spectrometry (16, 52) and were confirmed in the present study by coelution with authentic standards.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Metabolism of bisbenzamidoximes (compound 1, 3, 5, or 7) by rat liver homogenate 9,000 × g supernatant fraction. Homogenates containing 167 µM diamidoxime as substrate plus cofactor solution were incubated for 30 min at 37°C, then assayed by HPLC as described in Materials and Methods. Compound structures are shown in Table 1. IS, internal standard.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Time course of reduction of bisbenzamidoxime compound 1 to its monoamidoxime-monoamidine product (compound 9) by rat liver microsomes () and the postmitochondrial supernatant fraction (). Reaction conditions and the HPLC assay were as described for Fig. 1 and in Materials and Methods.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Metabolism of the bisbenzamidoxime compound 1 by 9,000 × g supernatants from homogenates of rat liver, kidney, lung, and heart. Incubation conditions were as described for Figure 1. Compound structures are shown in Table 1.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Metabolism of bisbenzamidoximes by intact BRL 3A hepatocytes in vitro. Cells cultured in Ham's F-12 medium containing 5% fetal bovine serum and 10 µM diamidoxime substrate were incubated for 24 h at 37°C under 5% CO2. The extracellular medium was extracted and assayed by HPLC as described for Fig. 1. Compound structures are shown in Table 1.

(ii) Metabolism of diamidoximes that lack anti-Pneumocystis activity. The novel bisbenzamidoxime compounds 11 and 13, the bisbenzimidazole diamidoximes 15 and 17, and the carbazole compound 19 had little or no activity orally or intravenously against Pneumocystis, even though the corresponding diamidines were very active intravenously (Table 1). Lack of activity is not associated with inability to be metabolized in vitro. Incubation of diamidoxime 15 with rat liver homogenates produced two new peaks, one that coeluted with the authentic standard for diamidine 16 and a larger peak with a relative retention time consistent with that predicted for the corresponding monoamidoxime (Fig. 5C). Similar results were obtained with diamidoximes 11 (Fig. 5A), 13, 17, and 19 (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Metabolism of diamidoximes that lack activity against Pneumocystis. (A and C) Metabolism by rat liver homogenate supernatant fractions as described for Fig. 1; (B and D) metabolism by intact BRL 3A hepatocytes as described for Fig. 4. Compound structures are shown in Table 1.